Iron plays critical roles in cellular metabolism, human health and disease. Fe/S clusters and hemes are synthesized in mitochondria and thus much cellular Fe is imported into this organelle. One aim of this translational research program is to elucidate the Fe-associated players involved in these processes, as this will provide new mechanistic insights into cellular Fe metabolism and help develop new strategies to treat Fe- related diseases. A great challenge in Fe trafficking is to determine whether low-molecular-mass (LMM) Fe complexes participate. Using a liquid chromatography system with an on-line ICP-MS, we have detected numerous LMM Fe complexes in yeast and human Jurkat cells. We are particularly interested in cytosolic Fe species that are imported into mitochondria, and in mitochondrial Fe species that are used for Fe/S cluster and/or heme biosynthesis. We will characterize and identify these LMM Fe complexes using Mossbauer (MB), EPR, mass spectrometry, NMR and X-ray absorption spectroscopy. We will probe the cellular functions of these species and identify associated Fe pathways leading into and operating within mitochondria. We will use WT cells grown under different conditions, and various genetic strains in which Fe metabolism has been altered. Fe trafficking will also be examined on the systems biology level using an ensemble of interrelated spectroscopic probes centered on MB spectroscopy. The distribution of Fe in WT and genetically modified cells and their organelles will be used to address the mechanism of Fe trafficking. We will examine strains that either accumulate or deplete Fe from the cytosol or mitos. We will characterize the mechanism of Fe/S cluster and heme biosynthesis by developing an assay for these processes using intact isolated mitos. We will determine whether a MB-detectable pool of mitochondrial FeII is used for Fe/S cluster and/or heme biosynthesis, and determine the function of each component of the pool. We will evaluate whether Fe is exported from mitochondria. We will investigate Fe trafficking in human cells that have been genetically modified to allow RNAi knockdowns and overexpressions of genes involved in Fe metabolism. We will knock- down frataxin and examine the phenotype that develops over time and with the extent of knock-down at long times. We will determine the order in which various Fe-accumulation characteristics develop and establish a mechanism for the genesis of the resulting diseased state. Other genes involved in Fe metabolism and disease will be knocked-down and/or overexpressed. Finally, we will dedicate a portion of our MB time to collecting and interpreting spectra of samples from other NIH-supported labs who study cellular Fe metabolism. Relevance of this research to public health. Numerous iron-associated diseases are related to problems in transporting iron into different parts of the cell. Iron often accumulates in the mitochondria, generating very small iron particles and highly reactive oxygen species that damage cells. Analogous diseased states will be recreated in yeast and human cells, and studied using sophisticated biophysical and bioanalytical methods. 1